Functional 3D Human Liver Bud Assembled from MSC-Derived Multiple Liver Cell Lineages

The severe shortage of donor liver organs requires the development of alternative methods to provide transplantable liver tissues such as stem cell-derived organoids. Despite several studies describing the generation of vascularized and functional liver tissues, none have succeeded in assembling human liver buds containing hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs). Here, we report a reproducible, easy-to-follow, and comprehensive self-assembly protocol to generate three-dimensional (3D) human liver buds from naïve mesenchymal stem cells (MSCs), MSC-derived hepatocytes, and HSC- and LSEC-like cells. By optimizing the ratio between these different cell lineages, the cell mixture self-assembled into 3D human liver buds within 72 h in vitro, and exhibited similar characteristics with early-stage murine liver buds. In a murine model of acute liver failure, the mesenteric transplantation of self-assembled human liver buds effectively rescued animal death, and triggered hepatic ameliorative effects that were better than the ones observed after splenic transplantation of human hepatocytes or naïve MSCs. In addition, transplanted human liver buds underwent maturation during injury alleviation, after which they exhibited a gene expression profile signature similar to the one of adult human livers. Collectively, our protocol provides a promising new approach for the in vitro construction of functional 3D human liver buds from multiple human MSC-derived hepatic cell lineages; this new technique would be useful for clinical transplantation and regenerative medicine research.